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Results for “Functional characterization”

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At least 469 records · Page 26Linked to original sources

PubMed2003

Functional characterization of human mesenchymal stem cell-derived adipocytes.

The function of adipocytes derived from human mesenchymal stem cells (hMSC) was investigated for the first time in hMSC from fetal liver (FL) and adult bone marrow (BM) and compared with preadipocytes from human subcutaneous adipose tissue differentiated according to adipocyte-specific protocols. FL- and BM-derived adipocytes displayed both morphological and functional characteristics of mature adipocytes including specific intracellular signaling pathways for tumor necrosis factor-alpha, catecholamine-regulated lipolysis as well as secretion of adiponectin and leptin. Similar to differentiated preadipocytes, hMSC adipocytes displayed lipolytic effects mediated by beta-adrenoceptors and antilipolytic effects mediated by the alpha 2A-adrenoceptor (alpha 2A-AR) and expressed proteins with a pivotal role in human lipolysis, including beta 2-AR, alpha 2A-AR, and hormone-sensitive lipase. We conclude that hMSC-derived adipocytes are morphologically and functionally similar to preadipocytes and display an intact lipolytic signaling pathway and endocrine function. These systems could be of great value in adipocyte research as a renewable source of adipocytes.

Adipocytes↗

PubMed2004

Functional characterization of genetic polymorphisms identified in the human cytochrome P450 4F12 (CYP4F12) promoter region.

The human cytochrome CYP4F12 has been shown to be active toward inflammatory mediators and exogenous compounds such as antihistaminic drugs. In the present study, we report the first investigation of polymorphisms in the human CYP4F12 gene. A screening for sequence variations in the 5'-flanking region was performed by a Polymerase Chain Reaction-Single Strand Conformational Polymorphism (PCR-SSCP) strategy, using DNA samples from 53 unrelated French individuals of Caucasian origin. Several polymorphisms were identified, comprising a large deletion located in intron 1 (CYP4F12*v1), two isolated substitutions -402G>A (CYP4F12*v3) and -188 T>C (CYP4F12*v4) and nine combined mutations, -474T>C, -279A>C, -224A>G, -173G>A, -145C>G, -140T>C, -126T>C, -56T>C, and -21T>G (CYP4F12*v2). Considering the nature and location of the polymorphisms characterizing the CYP4F12*v1 and *v2, the functional relevance of those two allelic variants was further examined by transfecting different cell lines with constructs of the related region of the CYP4F12/luciferase reporter gene. Both alleles lead to a significant decrease of CYP4F12 gene expression in HepG2 cell line and, therefore, are likely to determine interindividual differences in CYP4F12 gene expression.

Alleles↗

PubMed2002

Intrahepatic and circulating HLA class II-restricted, hepatitis C virus-specific T cells: functional characterization in patients with chronic hepatitis C.

To compare the functional features of circulating and intrahepatic hepatitis C virus (HCV)-specific CD4+ T cells in chronic HCV infection, peripheral blood and liver-infiltrating lymphocytes from 29 patients with chronic hepatitis C were stimulated with structural and nonstructural HCV proteins to produce antigen-specific T-cell lines and clones. Antigen specificity, fine specificity, phenotype, cytokine production, and T-cell receptor (TCR)-vbeta chain expression were analyzed. The results indicate a hierarchy of stimulatory capacity by the different HCV proteins, core being the antigen most frequently recognized by CD4+ intrahepatic lymphocytes, followed by NS4 and NS5. The CD4 response was directed simultaneously against different HCV proteins in individual patients, but fine-specificity analysis indicated that the response was generally focused on a limited number of immunodominant epitopes. Although the narrowly focused nature of this response may favor the emergence of escape mutations, this event was not observed by following-up over time the sequence of 2 epitopes strongly immunodominant for intrahepatic CD4 cells of a patient with chronic HCV infection. In conclusion, simultaneous analysis of peripheral blood and intrahepatic CD4 cells in the same patients indicated a predominant Th1 profile of HCV-specific CD4 cells and suggests a specific compartmentalization of virus-specific T cells into the liver.

Adult↗

PubMedDate not supplied

Functional characterization of P2X3 receptors fused with fluorescent proteins.

P2X receptor function in the CNS is poorly understood, and currently available data are partly inconsistent. In the presented study, we investigated P2X3 receptors stably expressed in HEK293 cells. Non-stationary noise analysis of whole cell currents and rapid ATP application through flash photolysis allowed for assessing the single channel conductance (6.6 pS) and the fast activation kinetics of the receptor (20 ms). The characteristics of channel desensitization and pharmacological properties matched previous findings. The properties of wild type receptors were compared with P2X3 constructs carrying a fluorescent tag (ECFP or DsRed2) at the C-terminus. These fluorescently labeled subunits formed functional receptors, with neither the affinity of the ligand binding site nor channel properties (ion selectivity, gating kinetics, single channel conductance) differing from wild type. We conclude that both fusion proteins tested here are suitable for generating transgenic mice, which can be expected to promote understanding of the physiological role of P2X3 receptors in CNS signaling.

Cell Line↗

PubMed1981

Precursors of murine B lymphocytes. Physical and functional characterization, and distinctions from myeloid stem cells.

The emergence of functional B cells was monitored in irradiated or unirradiated CBA/N recipients of either adult bone marrow or fetal liver from CBA/HT6T6 donors. The cells that are primarily responsible for the generation of B lymphocytes, at least during the first 6 wk, are rapidly sedimenting (4.5-6 mm/h), lack surface immunoglobulin, and are found in both the adult bone marrow and the fetal liver from day 12 onward. These pre-B cells are distinct from the colony-forming unit spleen (CFU-s) as demonstrated by the following criteria: (a) absence from yolk sac (19), (b) lack of correlation between CFU-s number and the ability to generate B cells in fetal liver populations of different ages of gestation, and (c) hybridoma antibodies that significantly inhibited B cell reconstitution but have no effect on CFU-s numbers. The antigen detected by this antiserum is present on both the fetal liver and bone marrow B cell progenitor, although its expression is not restricted to the B lineage. The pre-B cells that we monitor are not homogeneous, however, as both physical and functional differences are found. These observations reinforce our thesis that committed progenitor cells for the humoral immune system are formed early in development and thereafter constitute the major precursor pool for the generation of B lymphocytes.

Age Factors↗

PubMed2000

Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIalpha.

To investigate the function of a DNA topoisomerase III enzyme in Caenorhabditis elegans, the full-length cDNA of C.elegans DNA topoisomerase IIIalpha was cloned. The deduced amino acid sequence exhibited identities of 48 and 39% with those of human DNA topoisomerase IIIalpha and Saccharomyces cerevisiae DNA topoisomerase III, respectively. The overexpressed polypeptide showed an optimal activity for removing negative DNA supercoils at a relatively high temperature of 52-57 degrees C, which is similar to the optimum temperatures of other eukaryotic DNA topoisomerase III enzymes. When topoisomerase IIIalpha expression was interfered with by a cognate double-stranded RNA injection, pleiotropic phenotypes with abnormalities in germ cell proliferation, oogenesis and embryo-genesis appeared. These phenotypes were well correlated with mRNA expression localized in the meiotic cells of gonad and early embryonic cells.

Animals↗

PubMed1985

Antigenic profile and functional characterization of human peritoneal macrophages.

The antigenic profile and the functional properties of human peritoneal macrophages have been analyzed by using a panel of monoclonal antibodies (MoAb) and functional assays. All peritoneal macrophages were stained by the anti-class I HLA MoAb Q6/64. Between 40 and 100% of the cells were stained by the anti-HLA-DR + DP MoAb Q2/80, Q5/6, and Q5/13; approximately 80% of the cells were stained by the anti-HLA-DQ MoAb BT3.4, and about 95% were stained by the anti-macrophage MoAb OKM1. Peritoneal macrophages were not stained by the anti-dendritic cells MoAb Ki-M4 or by MoAb to T cell subsets, although all of the MoAb were reactive with the appropriate substrates. More than 60% of the cells expressed Fc receptors and C3 receptors, and displayed phagocytic activity. Peritoneal macrophages were effective in stimulating autologous and allogeneic lymphocytes and in presenting soluble antigens to T cells. These reactions were blocked by the anti-HLA-DR + DP MoAb Q5/13, but were not affected by the anti-dendritic cells MoAb Ki-M4 or by the anti-class I HLA MoAb Q6/64. These results suggest that human peritoneal macrophage preparations, without detectable contamination with dendritic cells, can induce proliferation of autologous and allogeneic T cells, and that class II HLA antigens play a significant role in these phenomena.

Antigen-Presenting Cells↗

PubMed1994

[Phenotypic and functional characterization of human prothymocytes].

We report herein the phenotypic and functional analysis of human bone marrow and thymus derived early T cells. Commitment to T cell lineage is acquired during CD7 antigen expression by CD34+ precursors in human bone marrow and before thymus colonization. Early thymocytes show similar phenotypic characteristics as bone marrow T cells. They rapidly acquire CD4 before the dual expression of CD4 and CD8. Their expansion and differentiation is regulated by two major factors: thymic stroma and cytokines produced by these stroma cells or by thymocytes themselves. Among cytokines, IL1 and sCD23 produced by thymic epithelial cells support in vitro early T cell development.

Adult↗

PubMed2002

Functional characterization of CitM, the Mg2+-citrate transporter.

The CitM transporter from Bacillus subtilis transports citrate as a complex with Mg2+. In this study, CitM was functionally expressed and characterized in E. coli DH5a cells. In the presence of saturating Mg2+ concentrations, the Km for citrate in CitM was 274 mM, similar to previous studies using whole cells of B. subtilis. CitM has a high substrate specificity for citrate. Other di- and tricarboxylic acids including succinate, isocitrate, cis-aconitate and tricarballylic acid did not significantly inhibit the uptake of citrate in the presence of Mg2+. However, CitM accepts complexes of citrate with metal ions other than Mg2+. The highest rate of citrate transport was seen in the presence of Mg2+, followed in order of preference by Mn2+, Ba2+, Ni2+, Co2+ and Ca2+. Citrate transport by CitM appears to be proton coupled. The transport was inhibited in transport buffers more alkaline than pH 7.5 and not affected by pH at acidic values. Transport was also inhibited by ionophores that affect the transmembrane proton gradient, including FCCP, TCC and nigericin. Valinomycin did not affect the uptake by CitM, suggesting that transport is electroneutral. In conclusion, the cloned CitM transporter from B. subtilis expressed in E. coli has properties similar to the transporter in intact B. subtilis cells. The results support a transport model with a coupling stoichiometry of one proton coupled to the uptake of one complex of (Mg2+-citrate)1-.

Bacillus subtilis↗

PubMed1999

Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae.

BACKGROUND: Porins are channel-forming membrane proteins that confer solute permeability to the outer membrane of Gram-negative bacteria. In Escherichia coli, major nonspecific porins are matrix porin (OmpF) and osmoporin (OmpC), which show high sequence homology. In response to high osmolarity of the medium, OmpC is expressed at the expense of OmpF porin. Here, we study osmoporin of the pathogenic Klebsiella pneumoniae (OmpK36), which shares 87% sequence identity with E. coliOmpC in an attempt to establish why osmoporin is best suited to function at high osmotic pressure. RESULTS: The crystal structure of OmpK36 has been determined to a resolution of 3.2 A by molecular replacement with the model of OmpF. The structure of OmpK36 closely resembles that of the search model. The homotrimeric structure is composed of three hollow 16-stranded antiparallel beta barrels, each delimiting a separate pore. Most insertions and deletions with respect to OmpF are found in the loops that protrude towards the cell exterior. A characteristic ten-residue insertion in loop 4 contributes to the subunit interface. At the pore constriction, the replacement of an alanine by a tyrosine residue does not alter the pore profile of OmpK36 in comparison with OmpF because of the different course of the mainchain. Functionally, as characterized in lipid bilayers and liposomes, OmpK36 resembles OmpC with decreased conductance and increased cation selectivity in comparison with OmpF. CONCLUSIONS: The osmoporin structure suggests that not an altered pore size but an increase in charge density is the basis for the distinct physico-chemical properties of this porin that are relevant for its preferential expression at high osmotic strength.

Bacterial Proteins↗

PubMed1991

Structure, expression and in vitro functional characterization of a novel RNA binding zinc finger protein from Xenopus.

Large multigene families of zinc finger proteins are expressed in vertebrates. One way of approaching their function is to characterize their structure, expression and biochemical properties. XFG 5-1 is a Xenopus zinc finger protein which is widely transcribed in oocytes, embryos and adult tissues. It carries a novel, non-finger repeat structure, which is common to a subfamily of Xenopus zinc finger proteins. The bacterially expressed protein exhibits specific RNA homopolymer binding activities with the zinc finger domain being sufficient for this ability. These findings suggest that XFG 5-1 serves a general biological function involving its RNA binding capacity.

Amino Acid Sequence↗

PubMed2006

Two factor H-related proteins from the mouse: expression analysis and functional characterization.

Complement factor H-related (FHR) proteins display structural and functional similarities to each other and to the complement regulator factor H (FH). FHRs have been identified in various species, including human, rat, and the fish barred sand bass. As mice provide a useful model system to study the physiological role of FHRs in vivo, we aimed at characterizing murine FHR proteins. Two putative FHRs of approximately 100 and 38 kDa were detected in mouse plasma using FH-specific antiserum. In a liver cDNA library, three murine FHR-encoding transcripts were identified. Two clones code for related FHR proteins termed FHR-C and FHR-C_v1, which in secreted form are composed of 14 and 13 short consensus repeat (SCR) domains, homologous to SCRs 6-17 and 19-20 of FH. The third transcript, FHR-B, is derived from a separate gene and codes for a secreted protein composed of five SCR domains. FHR-B displays homology to SCRs 5-7 and 19-20 of FH. Expression of FHR-B in various tissues was analyzed by real-time polymerase chain reaction and was identified at high levels in liver, kidney and heart. In liver, FHR-B transcript level was even higher than that of FH. In addition, FHR-B was expressed as a recombinant 37-kDa protein, and this recombinant FHR-B interacted with the ligands heparin and human C3b. Using mouse plasma, the native presumptive FHR proteins were also analyzed in binding assays. In summary, we identify two FHR proteins in mice and for the first time characterize a murine FHR as a heparin- and C3b-binding protein.

Amino Acid Sequence↗

PubMed2000

Functional characterization of five constitutively activating thyrotrophin receptor mutations.

OBJECTIVE: Gain of function mutations of the thyrotrophin receptor (TSHR) affect several functional characteristics, such as cAMP and inositol phosphate (IP) accumulation, cell surface expression and TSH affinity. In this study we compared five constitutively activating TSHR mutations, four receptors with a point mutation (S505N, L629F, I630L, V656F) and a nine amino acid (aa) deletion mutant (aa positions 613-621) for these functional parameters in parallel transfection experiments. METHODS: The wild-type TSHR (wt) and TSHRs containing the mutations S505N, L629F, I630L, V656F and the deletion 613-621 (all cloned in the expression vector pSVL) were transiently expressed in COS-7 cells in parallel experiments. Forty-eight hours after transfection the basal and stimulated cAMP and inositol phosphate accumulation as well as the cell surface expression (by FACS and ELISA), KD-values and TSHR down regulation by different stimuli were determined. RESULTS: In contrast to the very different values for specific constitutive activity (sca) (ranging from 7.5 to 100.3-fold wt) and very different levels of receptor cell surface expression (11-94% wt level) the basal cAMP accumulation determined in transfected COS-7 cells was surprisingly uniform (6.5-8.0 over wt basal). None of the point mutated receptors constitutively activates the phospholipase C cascade. In contrast the deletion 613-621 mutant showed constitutive activity for the IP pathway with a twofold increase in basal IP accumulation compared to the wild type TSHR. All investigated TSHR-mutants showed a TSH-stimulated receptor down-regulation, which seems to be independent of the phospholipase C pathway. CONCLUSIONS: The uniform basal cAMP values in spite of the large variation in specific constitutive activity values suggest that the COS-7 cell overexpression system used for the in vitro characterization is partly regulated. This regulation is most likely due to receptor down regulation. The TSHR deletion mutant (613-621) showed a constitutive activity for both the Galphas and the Galphaq/11 pathways. The TSH-mediated IP-stimulation by this mutant contrasts with its unresponsiveness to TSH for cAMP accumulation and therefore supports the model of different active conformations of the TSHR.

Animals↗

PubMed2001

Molecular and functional characterization of genes encoding horse MHC class I antigens.

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.

Amino Acid Sequence↗

PubMed1984

Functional characterization of human alloreactive T cell clones.

We have investigated the functional activity of alloreactive T cell clones, generated by limiting dilutions of MLC blasts. Clones displaying helper, suppressor, or cytotoxic functions (against Class I or Class II HLA antigens) were identified, using proliferative, 51Cr-release, and hemolytique-plaque induction assays. To determine whether functionally distinct clones share determinants that trigger the AMLC reaction we have tested the capacity of selected clones to cross-stimulate each other in secondary AMLC. Our data suggest that AMLC stimulatory determinants are related to common structure antigens of the T cell receptor for alloantigen, since helper clones, for example, (TLC #19) can induce memory responses to suppressor (TLC #209) as well as cytotoxic (TLC #207) clones and vice versa. The AMLC system seems well suited for studying network regulations within the T cell compartment.

B-Lymphocytes↗

PubMed1997

Genomic organization and functional characterization of the mouse GalR1 galanin receptor.

Galanin mediates diverse physiological functions in digestive, endocrine, and central nervous systems through G-protein-coupled receptors. Two galanin receptors have been cloned but the gene structures are unknown. We report genomic and cDNA cloning of the mouse GalR1 galanin receptor and demonstrate that the coding sequence is uniquely divided into three exons encoding the N-terminal portion through the fifth transmebrane domain, the third intracellular loop, and the sixth transmembrane domain through the C-terminus. Functional analysis of the encoded cDNA revealed active ligand binding and intracellular signaling. The expression is detected in brain, spinal cord, heart and skeletal muscle.

Amino Acid Sequence↗

PubMed2001

Preparation and functional characterization of thylakoids from Arabidopsis thaliana.

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O(2) and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate.

Journal Article↗

PubMed2004

Proteomic and functional characterization of endogenous adiponectin purified from fetal bovine serum.

Adiponectin is a plasma protein exclusively secreted from fat tissue. Many recent pharmacological studies suggest that recombinant adiponectin has multiple therapeutic potentials for obesity-related metabolic disorders, including type 2 diabetes, dyslipidemia, insulin resistance and atherosclerosis. However, the physiological relevance of these findings remains to be further established. In the present study, we have purified endogenous adiponectin from fetal bovine serum and characterized its post-translational modifications and physiological functions in animal models. Endogenous bovine serum adiponectin consists predominantly of full-length proteins that form multiple oligomeric complexes, including trimers, hexamers and higher molecular species. Two-dimensional gel electrophoresis revealed that bovine serum adiponectin exists as multiple post-translationally modified isoforms with distinct molecular weight and isoelectric point. Further analysis using mass spectrometry and Edman degradation sequencing demonstrated that five conserved lysine residues (Lys 28, 60, 63, 72 and 96) within the collagenous domain of bovine adiponectin are hydroxylated and glycosylated by a glucosyl alpha(1-2)galactosyl group. Injection of endogenous bovine adiponectin into C57 mice potently decreased circulating glucose levels and enhanced lipid clearance after a high fat meal. Chronic administration of this protein for a period of two weeks significantly increased insulin sensitivity and glucose tolerance, and depleted hepatic lipid accumulation in high-fat fed mice. These results provide direct evidence that endogenous bovine adiponectin is a physiological hormone that can regulate lipid and glucose metabolism.

Adiponectin↗